Project Summary/Abstract Glaucoma is the leading cause of irreversible blindness and is projected to affect 112 million people worldwide by 2040. Women represent 59% of the glaucoma population, highlighting the need to understand if there are sex-specific risk factors for glaucoma. For example, early menopause increases the risk of developing glaucoma. Estrogen receptor polymorphisms in women are associated with ocular hypertension (an important causal risk factor for glaucoma). Moreover, menopause causes a 1-3 mmHg increase in intraocular pressure (IOP), while hormone therapy containing estrogen reduces IOP. These data suggest that the age of menopause, menopause and estrogen levels influence glaucoma risk or IOP, but the mechanism(s) is unknown. IOP is affected by many factors, but aqueous outflow resistance is the key determinant, which is primarily controlled by trabecular meshwork (TM) function. Outflow resistance is segmental with high and low flow regions, and segmental flow correlates with TM stiffness. Notably, menopause and estrogen levels affect the stiffness of many tissues, suggesting a possible mechanism by which altered estrogen levels affect IOP. These changes in segmental flow may be related to altered protein expression since menopause alters protein levels known to influence aqueous outflow resistance. Thus, it is hypothesized that menopause increases IOP by increasing aqueous outflow resistance through stiffening the TM in high and low flow regions, thus increasing a woman?s risk of developing glaucoma. This proposal addresses the hypothesis in an animal model of menopause by measuring IOP and aqueous outflow resistance (Aim 1), and regional TM stiffness using Atomic Force Microscopy (Aim 2). This proposal achieves these aims using several approaches: Experiment A employs a well-established model of menopause, ovariectomy (OVX), in young (age 3-4 months) and middle-aged (age 9-10 months) female Brown Norway rats, and physiological menopause in elderly female rats (age 18-19 months). Experiment B will determine if topical estrogen therapy mitigates the effects of menopause, and investigates if this therapy affects IOP, outflow resistance, and TM stiffness in elderly male rats (age 18-19 months). In all animals, IOP will be measured using rebound tonometry for 8 weeks, followed by measurement of outflow resistance (via iPerfusion) and regional TM stiffness (via atomic force microscopy) in one eye. Contralateral eyes will be used to assess: estrogen levels or structure and composition along the outflow pathway. Preliminary data support this hypothesis, showing that IOP and aqueous outflow resistance are increased after OVX. This proposal will build on these data and expects to demonstrate that menopause increases TM stiffness and that topical estrogen therapy prevents these changes. These findings will provide functional and mechanistic insight into the association between menopause and glaucoma, and the benefits of topical estrogen therapy as a treatment for glaucoma.